1. Field of the Invention
The present invention relates to a method for the solubilization of an otherwise insoluble protein expressed by cultivated cells.
Cultivated cells, especially bacteria, in particular Escherichia coli; are used industrially for the overproduction of proteins, especially of proteins which are encoded by cloned genes of other organisms. Satisfactory overexpression of proteins of commercial interest has succeeded in many cases, as demonstrated by the examples of interferon-.beta. and insulin. However, it has emerged that there may be problems with the overproduction as a result of the insolubility of overexpressed proteins. A review on E. coli is to be found in Biotechnology, 5 (1987) 883-890.
2. The Prior Art
The proteins are deposited as inclusion bodies which are visible under the electron microscope; cf., for example, Science, 215 (1982) 687-689. While these inclusion bodies have the advantage that the recombinant proteins are substantially protected from proteolysis; cf., for example, EMBO Journal, 3 (1984) 1429-1434, the isolation of recombinant proteins in solubilized or active form from these inclusion bodies or deposits is associated with great difficulties. Often urea, sodium dodecyl sulfate (SDS) or other denaturing agents are used to dissolve the proteins. These detergents, however, lead to an undesired denaturation of the proteins; cf., for example, the following reviews: Marston (1987), the purification of eukaryotic polypeptides expressed in Escherichia coli. In DNA Cloning, Vol. III, ed. D. Glover, pages 59-88, IRL Press, Oxford; PNAS, 80 (1983) 906-910 or PNAS, 82 (1985) 2354-2358. However, a renaturation is associated with losses of active material, in particular, as the renaturation is not, as a rule, complete. There are also cases in which the renaturation is no longer possible, so that it is necessary to forego the insoluble protein, and use only the soluble fraction as a source of protein cf., for example, Biotechnology, 5 (1987), 960-965.